ATP-binding cassette (ABC) proteins are an ancient class of membrane transporters, found throughout phylogeny in eubacteria, archezoa and metakaryota (Gros. P. et al. (1986) Cell 47:371-370; Chen, C. J. et al. (1986) Cell 47:381-389; Kuchler, K. et al. (1989) EMBO J. 8:3973-3984; Riordan, J. R. et al. (1989) Science 245:1066-1073; Hyde, S. et al. (1990) Nature 346:362-365). During evolution, ABC proteins have become specialized in uptake and secretion, intracellular transport, cell detoxification and signaling and translocate highly diverse compounds across cell membranes, such as ions, amphiphiles, sugars, peptides and proteins.
The diverse functions and substrate specificities are accomplished by a common protein architecture (Hyde, S. et al. (1990) Nature 346:362-365). Each of the 1-4 functional units of an ABC protein consists of a hydrophobic domain of six membrane spanning segments and a hydrophilic cytoplasmic domain which is able to bind ATP. Three 20-45 amino acid sequence motifs in the nucleotide binding folds (NBFs) are highly conserved among ABC transporters.
The ABC protein family includes yeast (STE6 gene product), bacterial (haemolysin transport protein; hisP, malK, oppD and pstB proteins which are involved in ATP-dependent transport of specific molecules through bacterial inner cell membrane) and mammalian proteins. Four mammalian family members are currently known (PMP70, MHC-linked transport protein, P-glycoprotein, and the cystic fibrosis transmembrane conductance regulator protein (CFTR)).
PMP70
The peroxisomal membrane protein (PMP70) is one of the major integral membrane proteins of rat liver peroxisomes (Kamijo, K. et al. (1990) J. Biol. Chem. 265(8):4534-4540). It is believed that PMP70 may be involved in an active transport process through the peroxisomal membrane. One of the proposed functions for PMP70 protein is the transport of acyl-CoA compounds across peroxisomal membrane. Peroxisomes are cellular organelles bounded by a single membrane, and are observed in almost all types of eucaryotic cells. Although the physiological significance of peroxisomes has remained elusive, diseases caused by a general dysfunction of peroxisomes, including Zellweger syndrome, infantile Refsum disease, hyperpipecolic acidaemia, and neonatal adrenoleukodystrophy, have recently been recognized (Schutgens, R. B. H. et al. (1986) Eur. J Pediatr. 144:430-440). The severe clinical manifestation of these diseases indicate the indispensable roles of mammalian peroxisomes.
In cells of patients with Zellweger syndrome, peroxisomes are morphologically absent (Goldfischer, S. et al. (1973) Science 182:62-64). Biogenesis of peroxisomes is apparently impaired in this disorder; the cause may be defects in the protein machinery of the peroxisomes (Santos, M. J. et al. (1988) Science 239:1536-1538). It has been further postulated that the primary biochemical lesion is at the level of the biosynthesis of a protein essential for the import of peroxisomal enzymes from the cytoplasm into the peroxisomes which utilizes adenosine triphosphate during import (Schutgens et al. (1986) Eur. J. Pediatr. 144:430-440; Imanaka, T. (1987) J. Cell Biol. 105:2915).
MHC-linked (TAP) Transport Proteins
The MHC-linked (TAP) transport proteins appear to be required for antigen processing and presentation (Deverson, E. V. (1990) Nature 348:738-740; Monaco, J. J. et al. (1990) Science 250:1723-1726; Spies, T. et al., (1990) Nature 348:744-747). This MHC-linked transport protein may deliver intracellularly degraded antigens to the endoplasmic reticulum for binding to the class I major histocompatability molecules (Spies and DeMars (1991) Nature 351:323-324). Several mutant cell lines have been described in the literature, human B lymphoblastoid cell line (LCL) mutant and mutant murine cell line RMA-S, which have lost the ability to form peptide-MHC complexes. Since antigen that is derived from the cytoplasm must cross a lipid bilayer in order to associate with the external portion of MHC class I molecules, the most likely defect in these cells may be an inability to translocate the antigen from the cytoplasm into the appropriate membrane bound compartment where this association normally takes place. The region of the MHC implicated in these mutations contains the genes for the MHC-linked transport protein.
P-glycoprotein
P-glycoprotein is primarily expressed at epithelial and endothelial surfaces (Thiebaut, F. et al., (1987) PNAS USA 84:7735-7738) and is assumed to play an essential role in absorption and/or secretion. P-glycoprotein is believed to have two distinct and independent functions. P-glycoprotein is an active transporter which pumps hydrophobic drugs out of cells, reducing their cytoplasmic concentration and therefore toxicity. Thus, one function of P-glycoprotein is to eliminate toxic metabolites or xenobiotic compounds from the body (Endicott and Ling (1989) Annu. Rev. Biochem. 58:137-171; Croop, J. M. et al. (1988) J. Clin. Invest. 81:1303-1309; Gottesman and Pastan (1988) J. Biol. Chem. 263:12163-12166; Van der Bliek and Borst (1989) Adv. Cancer Res. 52:165-203).
P-glycoprotein has also been associated with a volume-regulated chloride channel activity. It has been reported that expression of P-glycoprotein generates volumneregulated, ATP-dependent, chloride-sensitive channels, with properties similar to channels characterized previously in epithelial cells (Valverde, M. A. (1992) Nature 355:830-8330). Therefore, P-glycoprotein is also believed to be involved in nutrient absorption in intestinal villus or in the placental cells at the site of material-foetal exchange (Trezise, A. E. O. (1992) EMBO J. 11(12):4291-4303). Unlike CFTR channels which are regulated by cyclic, P-glycoprotein is volume regulated, (e.g., swelling induced activation).
Overexpression of P-glycoprotein confers the phenotype of multidrug resistance (mdr; Gros, P. et al. (1986) Nature 323:728-731) which may cause failure of chemotherapy in cancer (Goldstein, L. J. et al. (1989) J. Natl. Cancer Inst. 81:116-124). The selection and proliferation of drug-resistant tumor cells represents a major cause of failure in the chemotherapeutic treatment of human tumors. Tumors initially sensitive to a cytotoxic agent often recur and are resistant to a broad spectrum of chemotherapeutic drugs (Wittes and Golden (1986) Cancer Treat. Rep. 70:105-125). From the study of highly drug-resistant cell lines derived in vitro, it is generally agreed that a net decrease of the intracellular concentration of drug underlies the multidrug-resistant phenotype (Bhalla, K. et al. (1985) Cancer Res. 45:3657-3662). It is now believed that P-glycoprotein binds the drugs to which a mdr cell is resistant or collaterally sensitive (Busche, R. et al. (1989) Mol. Pharmacol. 35:414-421; Busche, R. et al. (1989) Eur. J. Biochem. 183:189-197) and hydrolyzes ATP (Hamada and Tsuruo (1988) J. Biol. Chem 263:1454-1458).
Cystic Fibrosis Transmembrane Conductance Regulator protein (CFTR)
The cystic fibrosis transmembrane conductance Regulator protein (CFTR) is a 1480 amino acid protein containing two membrane-spanning domains (MSDs), two nucleotide binding domains (NBDs) and a unique R domain, that functions as a chloride channel regulated by phosphorylation and by nucleoside triphosphates.
Cystic Fibrosis (CF) is the most common fatal genetic disease in humans (Welsh M. J. et al. in The Metabolic Basis of Inherited Diseases, Vol. III, pp. 3799-3876 (Scriver, C. R. et al. eds., McGraw-Hill, New York (1995)). Approximately one in every 2,500 infants in the United States is born with the disease. At the present time, there are approximately 30,000 CF patients in the United States. Despite current standard therapy, the median age of survival is only 26 years. Disease of the pulmonary airways is the major cause of morbidity and is responsible for 95% of the mortality. The first manifestation of lung disease is often a cough, followed by progressive dyspnea. Tenacious sputum becomes purulent due to colonization of bacteria. Chronic bronchitis and bronchiectasis can be partially treated with the current therapy, but the course is punctuated by increasingly frequent exacerbations of the pulmonary disease. As the disease progresses, the patient's activity is progressively limited. End-stage lung disease is heralded by increasing hypoxemia, pulmonary hypertension, and cor pulmonale.
The upper airways of the nose and sinuses are also involved by CF. Most patients develop chronic sinusitis. Nasal polyps occur in 15-20% of patients and are common by the second decade of life. Gastrointestinal problems are also frequent in CF; infants may suffer meconium ileus. Exocrine pancreatic insufficiency, which produces symptoms of malabsorption, is present in the large majority of patients with CF. Males are almost uniformly infertile and fertility is decreased in females.
Based on both genetic and molecular analyses, a gene associated with CF was isolated as part of 21 individual cDNA clones and its protein product predicted (Kerem, B. S. et al. (1989) Science 245:1073-1080; Riordan, J. R. et al. (1989) Science 245:1066-1073; Rommens, J. M. et al. (1989) Science 245:1059-1065)). European patent application publication number: 0 446 017 A1 describes the construction of the gene into a continuous strand, expression of the gene as a functional protein and confirmation that mutations of the gene are responsible for CF. (See also Gregory, R. J. et al. (1990) Nature 347:382-386; Rich, D. P. et al. (1990) Nature 347:358-362).
The protein product of the CF associated gene is called the cystic fibrosis transmembrane conductance regulator (CFTR) (Riordan, J. R. et al. (1989) Science 245:1066-1073). CFTR is a protein of approximately 1480 amino acids made up of two repeated elements, each comprising six transmembrane segments and a nucleotide binding domain. The two repeats are separated by a large, polar, so-called R-domain containing multiple potential phosphorylation sites. Based on its predicted domain structure, CFTR is a member of a class of related proteins which includes the multi-drug resistance (MDR) or P-glycoprotein, bovine adenyl cyclase, the yeast STE6 protein as well as several bacterial amino acid transport proteins (Riordan, J. R. et al. (1989) Science 245:1066-1073; Hyde, S. C. et al. (1990) Nature 346:362-365). Proteins in this group, characteristically, are involved in pumping molecules into or out of cells.
CFTR has been postulated to regulate the outward flow of anions from epithelial cells in response to phosphorylation by cyclic AMP-dependent protein kinase or protein kinase C (Riordan, J. R. et al. (1989) Science 245:1066-1073; Frizzell, R. A. et al. (1986) Science 233:558-560; Welsh, M. J. and Liedtke, C. M. (1986) Nature 322:467; Li, M. et al. (1988) Nature 311:358-360; Hwang, T-C. et al. (1989) Science 244:1351-1353; Anderson, M. P. and Welsh, M. J. (1992) Science 257:1701-1704.
Sequence analysis of the CFTR gene of CF chromosomes has revealed a variety of disease causing mutations (Cutting, G. R. et al. (1990) Nature 346:366-369; Dean, M. et al. (1990) Cell 61:863:870; and Kerem, B-S. et al. (1989) Science 245:1073-1080; Kerem, B-S et al. (1990) Proc. Natl. Acad. Sci. USA 87:8447-8451). Population studies have indicated that the most common CF mutation, a deletion of the 3 nucleotides that encode phenylalanine at position 508 of the CFTR amino acid sequence (.DELTA.F508), is associated with approximately 70% of the cases of cystic fibrosis. This mutation results in the failure of an epithelial cell chloride channel to respond to cAMP (Welsh and Smith (1993) Cell 73:1251-1254). In airway cells, this leads to an imbalance in ion and fluid transport. It is widely believed that this causes abnormal mucus secretion, and ultimately results in pulmonary infection and epithelial cell damage. In addition to the processing defect, the function of CFTR-.DELTA.F508 is decreased as indicated by a reduced P.sub.O (Dalemans, W. et al. (1991) Nature 354:526-528; Denning, G. M. et al. (1992) Nature 358:761-764). G551S, a mutation in NBD1, is correctly processed but has altered ATP-dependent channel regulation resulting in a reduced P.sub.O (Anderson, M. P. and Welsh, M. J. (1992) Science 257:1701-1704). R 117H, which contains a mutation in the membrane-spanning domain, is also correctly processed, but has altered ion conducting properties producing an overall decrease in function (Sheppard, D. N. et al. (1993) Nature 362:160-164).
To date, the primary objectives of treatment for CF have been to control infection, promote mucus clearance, and improve nutrition (Welsh M. J. et al. in The Metabolic Basis of Inherited Diseases, Vol. III, pp. 3799-3876 (Scriver, C. R. et al. eds., McGraw-Hill, New York (1995)). Intensive antibiotic use and a program of postural drainage with chest percussion are the mainstays of therapy. However, as the disease progresses, frequent hospitalizations are required. Nutritional regimens include pancreatic enzymes and fat-soluble vitamins. Bronchodilators are used at times. Corticosteroids have been used to reduce inflammation, but they may produce significant adverse effects and their benefits are not certain. In extreme cases, lung transplantation is sometimes attempted (Marshall, S. et al. (1990) Chest 98:1488).
Most efforts to develop new therapies for CF have focused on the pulmonary complications. Because CF mucus consists of a high concentration of DNA, derived from lysed neutrophils, one approach has been to develop recombinant human DNase (Shak, S. et al. (1990) Proc. Natl. Sci. Acad USA 87:9188). Preliminary reports suggest that aerosolized enzyme may be effective in reducing the viscosity of mucus. This could be helpful in clearing the airways of obstruction and perhaps in reducing infections. In an attempt to limit damage caused by an excess of neutrophil derived elastase, protease inhibitors have been tested. For example, alpha-1-antitrypsin purified from human plasma has been aerosolized to deliver enzyme activity to lungs of CF patients (McElvaney, N. et al. (1991) The Lancet 337:392). Another approach would be the use of agents to inhibit the action of oxidants derived from neutrophils. Although biochemical parameters have been successfully measured, the long term beneficial effects of these treatments have not been established.
Based on knowledge of the cystic fibrosis gene, three general corrective approaches (as opposed to therapies aimed at ameliorating the symptoms) are currently being pursued to reverse the abnormally decreased chloride secretion and increased sodium absorption in CF airways. Defective electrolyte transport by airway epithelia is thought to alter the composition of the respiratory secretions and mucus (Welsh M. J. et al. in The Metabolic Basis of Inherited Diseases, Vol. III, pp. 3799-3876 (Scriver, C. R. et al. eds., McGraw-Hill, New York (1995); Quinton, P. M. (1990) FASEB J. 4:2709-2717). Hence, pharmacological treatments aimed at correcting the abnormalities in electrolyte transport are being pursued. Trials are in progress with aerosolized versions of the drug amiloride; a diuretic that inhibits sodium channels, thereby inhibiting sodium absorption. Initial results indicate that the drug is safe and suggest a slight change in the rate of disease progression, as measured by lung function tests (Knowles, M. et al. (1990) N. Eng.J.Med. 322:1189-1194; App, E. (1990) Am. Rev. Respir. Dis. 141-605.) Nucleotides, such as ATP or UTP, stimulate purinergic receptors in the airway epithelium. As a result, they open a class of chloride channel that is different from CFTR chloride channels. In vitro studies indicate that ATP and UTP can stimulate chloride secretion (Knowles, M. et al. (1991) N. Eng. J. Med. 325-533). Preliminary trials to test the ability of nucleotides to stimulate secretion in vivo, and thereby correct the electrolyte transport abnormalities are underway.
As with all pharmacological agents, issues such as drug toxicity and dosing will be important in developing an appropriate pharmacological agent for treating CF. A more fundamental consideration with pharmacological approaches to CF therapy is whether the chloride channel activity associated with CFTR is the crucial property that leads to the disease state. The CFTR is an epithelial Cl.sup.- channel with novel structure and regulation (Welsh, M. J. et al. (1992) Neuron 8:821-829; Riordan, J. R. (1993) Annu. Rev. Physiol. 55:609-630). CFTR is composed of two membrane spanning domains which contribute to formation of the ion conducting pore and three cytoplasmic domains that regulate channel activity: Two nucleotide binding domains (NBDs), and the R domain. The presence of two NBDs confer a complex and poorly understood mechanism of regulation on channel activity. Phosphorylation of the R domain by cAMP-dependent protein kinase (PKA) is necessary, but not sufficient, for channel activity. Once the R domain has been phosphorylated, the NBDs must bind (Anderson, M. P. and Welsh, M. J. (1992) Science 257:1701-1704; Thomas, P. J. et al. (1992) J. Biol. Chem. 267:5727-5730; Ko, Y. H. et al. (1994) J. Biol. Chem. 269:14584-14588; Hartman, J. et al. (1992) J. Biol. Chem. 267:6455-6458; Travis, S. M. et al. (1993) J. Biol. Chem. 268:15336-15339) and probably hydrolyze (Anderson, M. P. and Welsh, M. J. (1992) Science 257:1701-1704; Hwang, T. C. et al. (1994) PNAS USA 91:4698-4702; Baukrowitz, T. et al. (1994) Neuron 12:473-482; Nagel, G. et al. (1992) Neuron 360:81-84) ATP in order to open. In addition, ATP hydrolysis may be required to close the channel (Hwang, T. C. et al. (1994) PNAS USA 91:4698-4702; Baukrowitz, T. et al. (1994) Neuron 12:473-482). Studies of the CFTR containing site-directed mutations suggest that the two NBDs do not have equivalent functions in channel regulation (Anderson, M. P. and Welsh, M. J. (1992) Science 257:1701-1704), and it has been proposed previously that hydrolysis of ATP at NBD1 opens the channel, while hydrolysis of ATP at NBD2 regulates closure. An important goal of CF research is to understand the function of CFTR and to use that knowledge to develop better treatments for the disease.
A second approach to curing cystic fibrosis, "protein replacement" seeks to deliver functional, recombinant CFTR to CF mutant cells to directly augment the missing CFTR activity. The concept of protein replacement therapy for CF is simple: a preparation of highly purified recombinant CFTR formulated in some fusogenic liposome or reassembled virus carrier delivered to the airways by instillation or aerosol. However, attempts at formulating a CF protein replacement therapeutic have met with difficulties. For example, CFTR is not a soluble protein of the type that has been used for previous protein replacement therapies or for other therapeutic uses. There may be a limit to the amount of a membrane protein with biochemical activity that can be expressed in a recombinant cell. There are reports in the literature of 10.sup.5 -10.sup.6 molecules/cell representing the upper limit (Wang, H. Y. et. al. (1989) J. Biol. Chem 264:14424), compared to 2000 molecules /second/cell being reported for secreted proteins such as EPO, insulin, growth hormone, and tPA.
In addition to limited expression capabilities, the purification of CFTR, a membrane bound protein, is more difficult than purification of a soluble protein. Membrane proteins require solubilization in detergents. However, purification of CFTR in the presence of detergents represent a less efficient process than the purification process required of soluble proteins. Other potential obstacles to a protein replacement approach include: 1) the inaccessibility of airway epithelium caused by mucus build-up and the hostile nature of the environment in CF airways; 2) potential immunogenicity ; and 3) the fusion of CFTR with recipient cells may be inefficient.
A third approach to cystic fibrosis treatment is a gene therapy approach in which DNA encoding CFTR is transferred to CF defective cells (e.g. of the respiratory tract). However, methods to introduce DNA into cells are generally inefficient. Since viruses have evolved very efficient means to introduce their nucleic acid into cells, many approaches to gene therapy make use of engineered defective viruses. However, viral vectors have limited space for accommodating foreign genes. For example, adeno-associated virus (AAV) although an attractive gene therapy vector in many respects, has only 4.5 Kb available for exogenous DNA. DNA encoding the full length CFTR gene represents the upper limit. Gene therapy approaches to CF will face many of the same clinical challenges as protein therapy.
Although there has been notable progress in developing curative therapies for CF based on knowledge of the gene encoding CFTR, the expressed protein product and mechanism of action, there are obstacles confronting every approach. New approaches for treating CF and other diseases or conditions associated inadequate or inappropriate function of ATP-binding cassette (ABC) proteins are needed.